10.1021/nn401661d.s002 Jung-uk Shim Jung-uk Shim Rohan T. Ranasinghe Rohan T. Ranasinghe Clive A. Smith Clive A. Smith Shehu M. Ibrahim Shehu M. Ibrahim Florian Hollfelder Florian Hollfelder Wilhelm T. S. Huck Wilhelm T. S. Huck David Klenerman David Klenerman Chris Abell Chris Abell Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single-Molecule-Counting Immunoassays American Chemical Society 2016 analyte molecules 2 orders prostate cancer Ultrarapid Generation 10 min femtoliter droplets enzyme molecule Femtoliter Microfluidic Droplets enzyme molecules reporter enzyme chemical reactions 46 fM microfluidic device prototype system biomolecular complexes 2016-02-19 02:59:36 Journal contribution https://acs.figshare.com/articles/journal_contribution/Ultrarapid_Generation_of_Femtoliter_Microfluidic_Droplets_for_Single_Molecule_Counting_Immunoassays/2393923 We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 10<sup>6</sup> per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.